The major long term-objective of this laboratory is to understand the role of plasma vitamin-binding proteins in vitamin metabolism. In laying hens a variety of vitamin-binding proteins are synthesized by the liver, secreted in relatively large quantities into the plasma, and deposited as vitamin-protein complexes in yolk by the ovary. Three hypotheses generated by this system guide our work and may also apply to fetal nutrition in mammals. 1) The distribution of vitamins and their deposition in cells is mediated by specific binding proteins. 2) The binding proteins are not recycled and thus the amount of vitamin deposited is limited stoichiometrically by the amount of binding protein. 3) Once a vitamin is bound to its binding protein, it is the protein, not the vitamin, which determines the tissue of uptake. These hypotheses will be tested for biotin in the laying hen and their applicability to biotin metabolism in mammals will be explored. These studies may help to understand certain classes of biotin-responsive carboxylase deficiency in humans. The specific aims of this proposal include the development of a highly sensitive assay for plasma biotin-binding protein. This assay will be used to measure plasma and yolk biotin-binding protein as a function of dietary biotin consumption. It will also be used to search for biotin-binding protein in the plasma of pregnant mammals and in milk. The plasma clearance times and tissue distribution of [125]-biotin binding protein will be determined and compared to that of other plasma proteins in laying hens. A receptor for biotin-binding protein will be sought in the vitellin membrane of the ovary. Purified yolk biotin-binding protein will be further characterized. Methods to remove biotin bound to the protein will be developed, and the structural changes associated with biotin-binding will be studied on the apoprotein.